Molecular basis of <scp>EWS</scp> interdomain self‐association and its role in condensate formation

Ewing sarcoma, the second most common pediatric bone and soft tissue cancer, is caused by aberrant fusion of the RNA-binding protein EWS (EWS) low-complexity domain (EWS^LCD) to the DNA-binding domain of the transcription factor friend leukemia integration 1 (FLI1). The resulting fusion, EWS::FLI1, directly interacts with and engages in a dynamic interplay with EWS that drives tumorigenesis and regulates the function of both proteins. While EWS^LCD is known to promote self-association, the role of the RNA-binding domains (RBDs) of EWS, which include arginine-glycine-glycine (RGG) repeat regions and a structured RNA-recognition motif (RRM), remains less well understood. Here, we investigate the interplay between EWS^LCD and RBDs using biomolecular condensation assays, microscopy, nuclear magnetic resonance (NMR) spectroscopy, and molecular simulations. Our studies reveal that RBDs differentially influence EWS^LCD condensate formation and suggest that electrostatics and polypeptide-chain length likely contribute to this interaction. NMR spectroscopy and molecular dynamics simulations further demonstrate that EWS^LCD and the central RNA-binding region, comprising the RRM and RGG2 domains, engage in transient, non-specific interactions that are broadly distributed across both regions and involve diverse residue types. Specifically, tyrosine, polar residues, and proline within EWS^LCD preferentially interact with arginine, glycine, and proline residues in the RBD. Atomistic simulations of EWS confirm that the full-length protein exhibits a similar interaction profile with conserved chemical specificity, supporting a model in which a network of weak, distributed interdomain contacts underlies EWS self-association. Together, these findings provide molecular insight into the mechanisms of EWS condensate formation and lay the groundwork for understanding how interdomain interactions regulate EWS and EWS::FLI1 function.